PROJECT SUMMARY/ABSTRACT Many cell surface proteins (CSPs) that are essential for neural development have been identified, but we still lack an overall understanding of the logic of the cell-cell interactions that program the assembly of neural circuits. Our long-term goal is to understand how cell-cell interactions mediated by CSPs program the assembly of the intricate synaptic patterns of nervous systems. Many years ago, it was proposed that in ?hard- wired? systems such as the fish optic tectum and the insect CNS and neuromuscular system, each neuron or neuronal type is labeled by ?identification tags? that control synaptic specificity, and that these tags are represented by specific CSPs called ?surface labels?. The original hypotheses predicted that surface labels that control synaptic specificity should be: 1) expressed on small subsets of neurons or muscles, 2) recognized by receptors whose expression is also restricted to small subsets of neurons (and might themselves be surface labels), 3) required for or influence the formation of specific synaptic connections, 4) encoded by families of related genes. We discovered a network of interacting CSPs that satisfies all of these criteria, using a new approach in which we selected proteins for in vivo analysis from a global in vitro interaction network. The Garcia group at Stanford and our group at Caltech generated an extracellular ?interactome? for all Drosophila immunoglobulin superfamily (IgSF) proteins, and found a subfamily of 21 2-Ig domain cell-surface proteins, the Dprs, that selectively binds to another subfamily of 9 3-Ig domain proteins, the DIPs. Each dpr and DIP gene is expressed by a small and unique subset of neurons, and mutations in these genes produce specific alterations in synaptic connectivity. The objectives of the present application are to define whether and how interactions between Dprs and DIPs constitute a ?connectivity code? that contributes to wiring specificity in the Drosophila larval neuromuscular system. The primary hypothesis underlying this application is that engagement of Dprs with their DIP partners provides information that can control synaptic targeting decisions. We plan to attain the objectives of this application through three specific aims. The first of these examines how Dpr-DIP interactions control formation of an axon branch of a specific motor neuron. The second analyzes how another DIP expressed on a single motor neuron controls innervation of its muscle target. The third creates tools for analysis of all Dprs and DIPs and identifies those expressed by specific motor neurons and muscles. The expected outcome of the proposed research will be the acquisition of new insights into the mechanisms by which interactions among CSPs control the specification of synaptic connections in a relatively simple model system. This will have a significant positive impact for human health by increasing our understanding of conserved mechanisms involved in nervous system development and disease in humans.